Novel Pharmacological Activity of Artesunate and Artemisinin: Their Potential as Anti-Tubercular Agents

Abstract

Tuberculosis is a major infectious disease that globally causes the highest human mortality. From this aspect, this study was carried out to evaluate novel pharmacological activities/effects of artesunate and artemisinin causing anti-tubercular activity/effects against Mycobacterium tuberculosis (Mtb). The anti-Mtb activities/effects of artesunate and artemisinin were evaluated using different anti-Mtb indicator assays, such as the resazurin microtiter assay, the Mycobacteria Growth Indicator Tube (MGIT) 960 system assay, and the Ogawa slant medium assay, as well as in vivo tests. Artesunate showed selective anti-Mtb effects by strongly inhibiting the growth of Mtb compared to artemisinin, and consistently induced anti-Mtb activity/effects by effectively inhibiting Mtb in the MGIT 960 system and in Ogawa slant medium for 21 days with a single dose; its minimum inhibitory concentration was 300 µg/mL in in vitro testing. Furthermore, artesunate demonstrated an anti-tubercular effect/action with a daily dose of 3.5 mg/kg in an in vivo test for four weeks, which did not indicate or induce toxicity and side effects. These results demonstrate that artesunate effectively inhibits the growth and/or proliferation of Mtb through novel pharmacological activities/actions, as well as induces anti-Mtb activity. This study shows its potential as a potent candidate agent for developing new anti-tuberculosis drugs of an effective/safe next generation, and suggests novel insights into its effective use by repurposing existing drugs through new pharmacological activity/effects as one of the substantive alternatives for inhibiting tuberculosis.

Keywords: MGIT 960 system; Ogawa slant medium; artesunate; isoniazid; susceptibility-test.

Figure 1

Experimental design for in vivo tests of artesunate and artemisinin. (A) The evaluation of in vivo effects; the rats were divided into a normal group (n = 5), positive control group (n = 5/isoniazid group) and experimental groups (artesunate and artemisinin groups, n = 5/each group). The rats were orally administered with a single dose of 5 mg/kg of isoniazid, and 3.5 mg/kg of artemisinin and artesunate, once a day for four weeks, after four weeks of Mtb infection, respectively. (B) The evaluation of in vivo toxicity; the rats were orally administered with two different doses (1.75 and 3.5 mg/kg) of artemisinin and artesunate, once a day for four weeks of the experiment, respectively, and additionally observed for seven days.

Figure 2

The in vitro anti-Mtb activity/effect of artesunate and artemisinin against Mtb H37Rv measured using the REMA. (A) The viability of Mtb determined by a change in color of the REMA; and (B) the quantification analysis of the REMA that indicates the Mtb inhibitory rate of the compounds. Mtb was incubated with different concentrations (9.375–300 µg/mL) of the compounds, respectively. The blue color and dotted lines show the anti-Mtb activity/effect, and the pink color indicates bacterial growth. (GC; growth controls containing no antibiotic, Blank; blank controls without inoculation, SD; standard reference drugs (INH; isoniazid, RIF; rifampicin, 1.25–5 µg/mL)). All results were expressed as mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05 was considered to be statistically significant.

Figure 3

The anti-Mtb effect of artesunate and artemisinin measured using the MGIT 960 system assay. Mtb was incubated with different concentrations (37.5–600 µg/mL) of the compounds in the MGIT 960 system device at 37 °C for three weeks, respectively. All results were expressed as mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05 was considered to be statistically significant.

Figure 4

The anti-Mtb effect of artesunate against the growth/proliferation of Mtb confirmed using the Ogawa slant assay. Mtb was incubated with different concentrations (150–600 µg/mL) of artesunate on 2% Ogawa slant medium, respectively. Bacterial growth colonies were observed after three weeks of incubation. The dotted line indicates the anti-Mtb activity/effects of artesunate against Mtb. (INH; isoniazid, RIF; rifampicin, EMB; ethambutol). The Ogawa slant medium assay was carried out three times independently.

Figure 5

The in vivo tests of artesunate and artemisinin. The rats (six weeks/female) were divided into a normal group (n = 5), positive control group (n = 5/isoniazid group) and experimental groups (artesunate and artemisinin groups, n = 5/each group). The rats were orally administered with a single dose of 5 mg/kg of isoniazid, and 3.5 mg/kg of artesunate and artemisinin, once a day for four weeks, after four weeks of Mtb infection, respectively. The rats were carefully observed during the experimental periods. * p < 0.05 was considered to be statistically significant.

Figure 6

Changes of the body weight in rats following administration of artemisinin and artesunate. (A) The groups administered artesunate; and (B) the groups administered artemisinin. The rats were divided into a normal group (n = 4) and experimental groups (n = 8/artesunate two groups, n = 8/artemisinin two groups, n = 4/each group). The rats were orally administered with two different doses (1.75 and 3.5 mg/kg) of artemisinin and artesunate, once a day for four weeks, respectively, and additionally observed for seven days. The results were expressed as a percentage of the normal group and presented as the mean ± S.D.